The primary goal of this proposal is to investigate the mechanism whereby respiratory syncytial virus (RSV) nonstructural protein 1 (NS1) modulates innate immunity and cell survival through the mitochondrial system following RSV infection, and to combine this knowledge with nanoparticle technology to develop new ways to prevent and treat RSV infections. Veterans commonly have some form of chronic lung disease and are especially susceptible to respiratory viral infections like those caused by RSV and influenza. RSV is an opportunistic human respiratory pathogen that causes bronchiolitis in infants and pneumonia in immunocompromised adults and the elderly with an estimated 64 million infections and 166,000 deaths annually worldwide. RSV is becoming more and more of a serious threat to aging veterans. Despite progress, the precise nature of the RSV-induced innate immune response remains unclear. Our preliminary evidence shows that the NS1 protein associates with mitochondria and is found in complex with the mitochondrial antiviral signaling (MAVS) protein in RSV-infected A549 epithelial cells and inhibits the signaling of retinoic acid-inducible gene-I (RIG-I) and MAVS occurring via the caspase activation and recruitment domain (CARD). This interferes with IFN-2 production which inhibits the innate immune response. NS1 also blocks programmed cell death (apoptosis) of infected cells and prevents loss of mitochondrial membrane potential, thus promoting cell (and viral) survival. These data have led to the hypothesis that RSV utilizes NS1 to attenuate RIG-I- MAVS-induced antiviral IFN-2 production and to modulate mitochondrial function for increasing cell survival. The study of the precise mechanisms underlying these processes is expected to lead to the discovery of new targets for preventing or limiting RSV infection. To test these hypotheses, the following specific aims are proposed. In the first aim, it is planned to examine the role of NS1 in attenuating RIG-I/MAVS signaling during RSV infection. This includes analysis of the role of NS1 in regulating MAVS, LGP2 and RIG-1 expression and in attenuation of IFN-2 response during RSV infection, mitochondrial localization of NS1 protein and structure-function relationships of NS1- MAVS interaction. The second aim will focus on determining whether the NS1- MAVS interaction is required to prevent premature apoptosis and intrinsic cell death. This also includes addressing whether the NS1-MAVS complex is associated with anti- or pro-apoptotic factors and modulates their expression on the mitochondrial membrane. In the third aim, it is planned to develop and test targeted chlipid (chitosan-lipid) nanoparticles as a therapeutic agent for RSV-induced lung disease. These chlipids encapsulate a plasmid that encodes siRNAs for LGP2 and NS1 and a peptide for MAVS (referred to as pLMNS1), each of which individually has been shown to significantly down- regulate RSV replication in human cells. Lastly, in the fourth aim it is proposed to evaluate targeted nanoparticle-encapsulated pLMNS1 for treating RSV disease in a mouse model. The proposed research will be conducted by an excellent group of investigators with a proven track record in RSV disease, apoptosis and nanoparticle technology. The results are expected to lead to the discovery of novel targets and to the initiation of preclinical studies of these targets against RSV lung disease.